Among a variety of ionization techniques applied to mass spectrometry, electrospray ionization (ESI) has evolved into a powerful and widely practiced tool for the analysis of high molecular weight biological molecules. The success of ESI in the analysis of biomolecules lies in the method's ability to extract fragile chemical species intact from solution in an ionized form, and transfer them to the gas phase for mass analysis. A unique characteristic of the electrospray (ES) ion source is the ability to form multiply-charged ions, which facilitates the analysis of extremely high molecular weight molecules with mass analyzers having relatively low nominal upper mass limits. Electrospray ionization methods have been extensively reviewed. See, for example, reviews by Banks, Jr. and Whitehouse in Methods in Enzymology, Vol. 270, 1996, pp. 486-519; and Smith, R. D., et al., Analytical Chemistry, Vol. 62, 1990, pp. 882-899. In an ES ion source, a liquid sample is introduced through a small bore tube that is maintained at several kilovolts at or near atmospheric pressure into a chamber containing a bath gas. A strong electrostatic field at the tip of the tube charges the surface of the emerging liquid generating Coulomb forces sufficient to overcome the liquid's surface tension and to disperse the liquid into a fine spray of charged droplets. After passing through the atmospheric-low pressure interface and desolvation region, ions are injected into a mass spectrometer. For analysis of complex samples, the multicharged ion formation characteristics of Electrospray Ionization Mass Spectrometry (ESI-MS) complicate mass spectral analysis, particularly for high mass biomolecules. Under the current understanding in the art, it is unclear why multicharged ion distributions observed in electrospray mass spectra are so different from the charge distributions of the corresponding ions in solution. For example, ESI mass spectra of positive ionized peptides or proteins are usually collected under pH conditions such that all or nearly all basic amino acid residues inside this peptide are be protonated with a probability extremely close to 1. Essentially, only ions with maximal possible charge are expected to exist in solution but ESI mass spectrum exhibits a wide distribution of multicharged ions. Since charge distributions of ions in solution are well established and since these distributions can be controlled by changes of the solution pH (properly controlling other experimental conditions), it would be highly valuable analytically to develop methods of extracting ions from solution while conserving their equilibrium solution charge distribution. The important property of biomolecules in solution is the isoelectric point, which is determined by the solution pH when the total charge of the biomolecule is zero. Using so called isoelectric focusing, it is possible to achieve good separations of biomolecules in gel electrophoresis techniques, where a difference in isoelectric points of about 0.01 is sufficient. Additional separation techniques for analysis of multicharged large ions would also be useful. Ion mobility is a technique of great interest as ion mobility resolving power increases proportionally to the square root of ion charge, yielding not only improved peak separation in the mobility cell but in addition, the mobility peak width may provide information about the ion charge state.
An IMS is typically composed of an ionization source, a drift cell, and an ion detector; examples of the latter include a sampling plate, an electron multiplier, or a mass spectrometer. Ion mobility spectrometry separates ions in terms of their mobility with reference to a drift/buffer gas by measuring the equilibrium velocity of the ions. When gaseous ions in the presence of a drift gas experience a constant electric field, they accelerate until a collision occurs with a neutral molecule. This acceleration and collision sequence is repeated continuously. Over time, this scenario averages out over the macroscopic dimensions of the drift tube to a constant ion velocity based upon ion size, charge, and drift gas pressure. The ratio of the velocity of a given ion to the magnitude of the electric field experienced by it is the ion mobility. In other words, the ion drift velocity (vd) is proportional to the electric field strength (E) where the ion mobility K=vd/E is a function of the ion volume/charge ratio. Thus IMS is a technique similar to mass spectrometry, having a separation component to it. The IMS technique is generally characterized as having high sensitivity with moderate separation power. Separation efficiency is compromised when “bands” of the various ions spread apart as opposed to remaining together in a tight, well-defined beam. This efficiency or resolving power for what is considered “classic” ion mobility (using uniform or quasi-uniform electric field to effect a separation due to the Einstein relationship between mobility coefficient and diffusion coefficient for ions for given ion charge) increases as the square root of applied voltage along mobility cell. This maximum voltage for a given length of mobility cell is restricted by the possibility of glow discharge and decomposition of ions due to heating from rapid velocities in the buffer gas. Increasing the buffer gas pressure does allow application of higher cell voltages and improved mobility resolving power.
Another possible analytical technique, using a new continuous flow technique for separation of gas-phase ions at atmospheric pressure, and referred to as high-field asymmetric waveform ion mobility spectrometry (FAIMS), has recently been described. (see R. W. Purves, R. Guevremont, S. Day, C. W. Pipich, M. S. Matyjaszczyk, Rev. Sci. Instrum. 69, 1094-4105 (1998); R. Guevremont, R. W. Purves, Rev. Sci. Instrum. 70, 1370-1383 (1999)). This technique is simply a further development of the cylindrical geometry case of the method implemented for the plane geometry and described earlier. (see I. A. Buryakov, E. V. Krylov, E. G. Nazarov, U. K. Rasulev Int. J. Mass Spectrom. Ion Processes 128, 143-148 (1993)). Adequate separation capability of this method for isomeric compounds was demonstrated. see D. A. Barnett, B. Ells, R. Guevremont, R. W. Purves “Separation of leucine and isoleucine by elecrtospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry”; J. Am. Soc. Mass Spectrom. 10, 1279-1284 (1999)). This approach is more suitable for coupling with continuous ionization methods such as electrospray. Its main difference from classic ion mobility spectrometry is focusing and recording of only one type of the ions from continuous ion flow for each time moment. All other ions are usually lost. The situation is the same as for all instruments of scanning type which may be adequate when the amount of the sample is not so important or when determination of only one or few known components is necessary. However, use of multi-beam ion pre-selection as proposed in the present invention partially overcomes this drawback and finds general use. Herein we describe the specific embodiment of the modified FAIMS for analysis of aerosol particles.
The combination of an ion mobility spectrometer (IMS) with a mass spectrometer (MS) is well known in the art. In 1961, Barnes et al. were among the first to combine these two separation methods. Such instruments allow for separation and analysis of ions according to both their mobility and their mass, which is often referred to as two dimensional separation or two dimensional analysis. Young et al. realized that an orthogonal time-of-flight mass spectrometer (oTOFMS) is the preferred mass spectrometer type to be used in such a combination because of its ability to detect simultaneously and very rapidly (e.g., with a high scan rate) all masses emerging from the mobility spectrometer. Their combination of a mobility spectrometer with an oTOFMS is herein referred to as an Ion Mobility-oTOFMS or IM-oTOFMS. This instrument comprised means for ion generation, a mobility drift cell, and an oTOFMS with a small orifice for ion transmission coupling the mobility cell to the oTOFMS.
Use of MS as a detector allows for resolution based on mass-to-charge ratio after separation based upon ion mobility. Shoff and Harden pioneered the use of Mobility-MS in a mode similar to tandem mass spectrometry (MS/MS). In this mode, the mobility spectrometer is used to isolate a parent ion and the mass spectrometer is used for the analysis of fragment ions (also called daughter ions) which are produced by fragmentation of the parent ions. Herein, this specific technique of operating a Mobility-MS is referred to as Mobility/MS, or as Mobility/TOF if the mass spectrometer is a TOFMS-type instrument. Other instruments and methods using sequential IMS/MS analysis have been described (see, e.g., McKight, et al. Phys. Rev., 1967, 164, 62; Young, et al., J. Chem. Phys., 1970, 53, 4295; U.S. Pat. Nos. 5,905,258 and 6,323,482 of Clemmer et al.; PCT WO 00/08456 of Guevremont) but none combine the instrumental improvements disclosed presently. When coupled with the soft ionization techniques and the sensitivity improvements realizable through use of the drift cell systems herein disclosed, the IMS/MS systems and the corresponding analytical methods of the present invention offer analytical advantages over the prior art, particularly for the analysis of macromolecular species, such as biomolecules.
The challenging issue when constructing an IMS-MS device is to achieve a high ion transmission from the mobility region into the MS region of the tandem instrument. It is at this interface that the earlier approaches of ion mobility technology using a linear field appear incongruous with the goal of maximizing ion throughput across the IMS/MS interface. The mobility section is operating at a pressure of typically between 1 mTorr and 1000 Torr whereas the MS is typically operating at pressures bellow 10−4 Torr. In order to maintain this differential pressure it is necessary to restrict the cross section of the opening that permits the ions to transfer from the mobility section to the MS section. Typically this opening cross section is well below 1 mm2. Hence it is desirable to focus the ions into a narrow spatial distribution before this interface transmission occurs. Another important property of the ion beam arriving into the MS is the divergence of this beam in the kinetic energy for ion motion in the plane orthogonal to the direction of their insertion into the MS. Ion beam energy divergence is the main factor responsible for the resolution properties of the mass spectra for orthogonal TOFMS. In 2004, Loboda U.S. Pat. No. 6,744,043 described several versions of using of radio frequency (RF) ion guide for focusing of ions inside the mobility cell. However, this approach is suitable for low pressure ion mobility separation not more than a few Torr. Furthermore, RF focusing of ions decreases with increasing of m/z of ions so this method has some important restrictions. As discussed herein, RF focusing of ions in interface region just after the exit orifice of the mobility cell and before the entrance orifice of TOFMS is free from these drawbacks.
H. H. Hill, in the late 1980's, developed methods for introducing large biomolecules from aqueous samples directly into IMS using electrospray ionization techniques. (see Hill, H. H.; and Eatherton, R. L., “Ion Mobility Spectrometry after Chromatography-Accomplishments Goals, Challenges”, J. Research of the National Bureau of Standards, Accuracy in Trace Analysis, 93(3), 1988, 425; see Shumate, C. B.; and Hill, H. H., “Coronaspray Nebulization and Ionization of Liquid Samples for Ion Mobility Spectrometry”, Analytical Chemistry, 61, 1989, 601. Recently, Hill and co-workers have interfaced a high resolution atmospheric pressure ion mobility spectrometer to a time-of-flight mass spectrometer and obtained rapid 2-D separations of amphetamines (Steiner, W. E.; Clowers, B. H.; Fuhrer, K.; Gonin, M.; Matz, L. M.; Siems, W. F.; Schultz, A. J.; and Hill, H. H., “Electrospray Ionization with Ambient Pressure Ion mobility Separation and Mass Analysis by Orthogonal Time-of-Flight Mass Spectrometry”, Rapid Commun. Mass Spectrom., 15, 2001, 2221-2226), PTH-amino acids (Steiner, W. E.; Clowers, B. H.; Hill, H. H., “Rapid Separation of Phenylthiohydantoin Amino Acids: Ambient Pressure Ion Mobility Mass Spectrometry (IMMS)”, Anal. and Bioanal. Chem., accepted October 2002), and chemical warfare degradation products (Steiner, W. E.; Clowers, B. H.; Matz, L. M.; Siems, W. F.; Hill, H. H., “Rapid Screening of Aqueous Chemical Warfare Agent Degradation Products: Ambient Pressure Ion Mobility Mass Spectrometry (IMMS)”, Anal. Chem., 2002, 74, 4343-4352). At the interface between the IMS and the TOF, collision-induced dissociation of mobility separated ions can be turned on and off by varying the interface voltage to provide an added dimension of analysis. This and other known approaches for coupling of electrospray ion source with IMS/MS all suffer from large losses of ions in all stages of their transport and some decreases in mobility resolving power due-to significant width of initial ion package formed by interruption (pulse-forming) of the continuous ion flow from the electrospray ion source. The typical sensitivity of these measurements is in the range of μM, which is far worse than that for typical non-IMS electrospray and matrix-assisted laser desorption ionization (MALDI) measurements. MALDI sensitivities in the femto-molar range are typical (a difference of up to nine orders of magnitude). As the continuous electrospray ion source direct is chopped (or pulsed) for introduction of the ion package into mobility cell only approximately 1% of the initial ion source production is utilized in the mobility cell. The relative time width of this ion package to the time between such introductions should be less than the inverse of expected mobility resolving power. Thus, increasing mobility resolving power would lead in this case to additional losses of ions and a further decrease in sensitivity. This pulse-forming condition is related to that with coupling of continuous ion source with TOFMS before the invention of orthogonal injection of ions into TOFMS. Herein, a method of ion injection into mobility cell is demonstrated which is free from the beam-chopping limitations of usual coaxial introduction of ions.
In 2004, Eriksson U.S. Pat. No. 6,683,302 described an electrospray ion source where heating of droplets emerging from the electrospray capillary under the influence of a strong electric field was provided by microwave energy directed between the spray tip and mass analyzer.
In 2003, Ranasinghe, et al. U.S. Patent Application 2003/0001090 described splitting the liquid flow from a separation device into two approximately equal streams and directing them into two ion spray sources; the first one producing positive ions and the second one producing negative ions. Two TOFMSs were used for recording of these positive and negative ions. In 2004, Van Berkel U.S. Pat. No. 6,677,593 described partial separation of ions in a liquid phase by applying electric or magnetic fields or their combination. Enriched positive ion flow is directed into one capillary whereas the flow with negative ions is sent through another capillary. Due to the large electric field near the tips of the capillaries during operation of the electrospray ion source from solution phases, charge distribution of ions are “spoiled” in the ion formation and extraction process.
In 2004, Berggren, et al. U.S. Pat. No. 6,797,945 described some versions of using piezoelectric formation of charged droplets for electrospray ion source. This approach may be promising for several reasons. ESI coupled with pulsed techniques of ion analysis in classic ion mobility spectrometers is simplified because it is possible to form droplets in controllable short time intervals. It is also appears to be important that droplets may be produced having well known and narrow size distributions. Berggren teaches that it is possible to get ions with less spread in their charges by applying less voltage to the tip of the capillary from where the droplets emerge. However, application of any voltage (to the piezoelectric element located inside investigated solution) may change, to some extent, the conditions for ion formation. Therefore, the charge distribution inside large ions of interest may still be changed from that in the solution at given pH and without additional influences.
An idea to mix microwave voltage for heating with quasi-periodic signal with frequency band 10-10000 kHz for splitting of combustion kernels in internal combustion engine was suggested in 1999 by Gordon, et al. U.S. Pat. No. 5,983,871.
In 2004, Apffel, et al. U.S. Pat. No. 6,797,946 described the nebulizing of solutions and ionization of the neutral species contained in the solutions by atmospheric pressure ionization (API) and atmospheric pressure chemical ionization (APCI) as well as suggesting orthogonal injection of resulting ions into the vacuum part of mass spectrometer. The described version of orthogonal injection of ions may be considered as a further development of the widely used approach for removing of large and low charged droplets from electrospray flow by a gas curtain. Some advantages of this approach may be expected: lower “curtain” gas flow as it is injected in the same direction as electrospray flow, and perhaps, some better sensitivity of measurement and less evaporated solvent flow inside mass spectrometer. However, Apffell nowhere suggests using gas counterflow, ion accumulation in traps, and pulse inserting of ions for analysis which are aspects of the present invention discussed herein.
In 2005, Takats, et al. U.S. Patent Application 2005/0029442 described ion spray from solution using increased speed (more than sound) of nebulizing gas flow assisted with voltage applied to the sample capillary. The experimental data were presented showing very narrow distribution of multicharged ions, sometimes showing reduction to one type of ion. Changes of average ion charge and peak width with applied voltage and the distance from the sample capillary tip to the input heated capillary for inserting ions into mass analyzer for different sample flows were measured. It was shown that ions with relatively low number of charges and low intensity may be detected for zero voltage applied to the sample capillary. The data given for nanoelectrospray for different spray voltages indicate more average charges for the same voltages after some onset voltage below which no ions are detected.
One issued U.S. patent and two pending U.S. patent applications of Schultz et al. (pending U.S. application Ser. No. 10/861,970, filed Jun. 4, 2004; pending U.S. application Ser. No. 11/231,448, filed Sep. 21, 2005; and U.S. Pat. No. 6,989,528) describe a system whereby massive cluster ions or massive cluster ions neubulized in a solvent may be impinged upon a surface both to liberate and ionize surface bound molecules or elements (SIMS) as well as simultaneously providing for nondestructive implantation of a portion of this droplet into the near surface region of a biopolymer which can thereafter be irradiated with a energetic particle source such as a laser (MALDI) for liberation of the molecules within the surface region. These U.S. patent applications are incorporated by reference as though fully described herein). A recently published variant of this approach was called Desorption Electrospray Ionization (DESI) (see Z. Takats, J. M. Wiseman, B. Gologan, R. Graham Cooks; Science Vol. 306, 15 Oct. 2004, pp 471-473). These techniques appears to be a useful tool for the investigation of a variety of surfaces of natural origin including in vivo analyses. The essence of these approaches involves directing the flow of solvent droplets acquired by nebulizer-assisted electrospray to the surface under investigation which is held under usual ambient conditions and insertion of the resulting flow from the surface into a mass spectrometer through an atmospheric pressure interface. Interesting experimental results were demonstrated including the mass spectrum from the finger of a person 50 min after taking 10 mg of the over-the counter antihistamine Loratadine (m/z 383/385). The corresponding peaks are clearly seen in the spectrum. It is stated in the paper that “changes in the solution that is sprayed can be used to selectively ionize particular compounds.” However use of high voltage applied to the solvent in the spraying capillary would change the conditions for formation of ions from the sample compared to those for initial solvent. Thus, for example, the control of pH in the solvent for producing of ions with corresponding charge distribution is impossible in this case as is the case for a typical electrospray ion source. A method free from this drawback is an aspect of the present invention.
Attempts to perform fast three dimensional separation of ions are also known. In 2001, Clemmer, et al. U.S. Pat. No. 6,323,482 described an approach whereby a quadrupole mass filter is located between mobility cell and time-of-flight instrument and is used for separation of non-resolved mobility peaks for providing collision-induced dissociation for selected ions. In 2003, also Clemmer U.S. Pat. No. 6,559,441 suggested the performance of two consecutive separations of ions before mass analysis due to two different molecular characteristics.
In 2004, Woods and Virgil, in U.S. Pat. No. 6,797,482, described the approach for high-resolution identification of solvent-accessible amide hydrogens in protein binding sites. Exchange in solution of “open” hydrogen atoms for heavy hydrogen atoms—tritium and deuterium—is used. Therefore, hydrogen atoms buried inside folded proteins are not exchanged. To reveal the corresponding amino acid residues with substituted and non-substituted H-atoms, proteolysis by special enzymes working under low temperature (close to 0° C.) and in strong acidic conditions (for pH about 2, 7) is used. Such low pH values and low temperatures significantly suppress isotopic exchange of H-atoms so it is possible to conserve information about initial structure of the protein in solution. Further HPLC separation is performed in such severe conditions for the same reason. The number of substituted H-atoms in different fractions is estimated by scintillator counting for the case of tritium exchange and mass spectrometry measurements for the case of deuterium exchange. The '482 patent gives a detailed overview of this field. It teaches that using mass spectrometry for solving these problems is restricted to overall determination of the number of substituted H-atoms for corresponding ions without further attempts to locate the sites having these atoms. Using the approach described therein, it is difficult to find locations of substituted H-atoms very precisely.
All of the above-referenced U.S. patents and published U.S. patent applications are incorporated by reference as though fully described herein.
Although much of the prior art resulted in improvements in ion production, focusing, separation, and in ion throughput from ion source to the mobility cell and to the mass spectrometer in tandem instruments, there is room for additional improvement in all these directions. The inventors describe herein a concept and designs of a new type electrospray ion source, multi-beam ion mobility and mass separations with multi-channel data recording which result in instrumental embodiments to provide improved ion production from investigated samples, their separation and measurements.